Acanthoatractis xinguensis n. gen., n. sp. (Nematoda: Cosmocercoidea: Atractidae) parasite of yellow-spotted Amazon river turtle, Podocnemis unifilis Troschel (Testudines: Podocnemididae) in Brazilian Amazon

Nematodes collected from the stomach of the yellow-spotted turtle Podocnemis unifilis Troschel, 1848 (Testudinidae) in the Brazilian state of Pará are assigned to a new genus, allocated to the family Atractidae (Cosmocerdoidea). Acanthoatractis n. gen. differs from all other genera of Atractidae based on the combination of the following morphological characters: cephalic extremity surrounded by eight bifurcated, wrench-shaped sclerotized structures arranged in a circle; oral opening encircled by two sclerotized pieces with pointed ends and a median portion with a pair of hooks; in males the larger (left) spicule is narrower in the middle third and the gubernaculum is absent. The type species, Acanthoatractis xinguensis n. gen., n. sp., has nine pairs of caudal papillae and a single papilla anterior to the cloacal lip. The new species is the seventh record of an atractid genus parasitizing P. unifilis.


Materials and methods
Three specimens of Podocnemis unifilis were collected from the Xingu River ("Volta Grande" region), municipality of Vitória do Xingu, Pará, Brazil, during a survey of helminths in freshwater turtles.Hosts were anesthetized by injection of 2% ketamine and posteriorly euthanized by ketamine overdosage.The organs of the gastrointestinal tract (stomach, small and large intestines) were carefully removed, isolated in Petri dishes, and examined under a LEICA EZ4 stereomicroscope (Leica Microsystems, Wetzlar, Germany).Nematodes were washed in saline solution (NaCl 0.9%) and heat-killed in 70% ethanol.For morphological and morphometric analysis, the nematodes were cleared in 20% Aman's lactophenol, following a protocol adapted from Amato et al. (1991), and examined using an Olympus BX41 microscope (Olympus, Japan) with a drawing tube.Measurements are in micrometers unless otherwise indicated and are presented as the range followed by the mean in parentheses.
We selected six specimens (three males and three females) for scanning electron microscopy (SEM) analyses.The nematodes were post-fixed in 1% OsO 4 , dehydrated in a graded ethanol series (30-100%), critical point dried with CO 2 , and placed on aluminum stubs using carbon tape and sputter coated with gold/palladium.We analyzed the specimens under a Vega3 microscope (TESCAN, Brno, Czech Republic) with an acceleration voltage of 10 kV in the Laboratory of Cellular Structural Biology (LBE) at the Federal University of Pará (UFPa).
The type-material was deposited in the Invertebrate Collection of the Helminthological Collection of Oswaldo Cruz Institute (CHIOC), Brazil.

Acanthoatractis Jesus and Melo n. gen
Diagnosis: Cosmocercoidea.Atractidae.Body medium-sized, tapering anteriorly, tail short and pointed with cuticle finely longitudinally striated.Lateral alae absent.Cephalic end surrounded by eight bifurcated sclerotized structures (two subdorsal pairs, two dorsolateral pairs, two subventral pairs, and two ventrolateral pairs) shaped like single-ended wrench, arranged in circle.These sclerotized structures equal, positioned on opposite sides in both transverse and longitudinal planes.Oral aperture small, terminal, and rectangular, surrounded by two sclerotized pieces (one ventral and one dorsal); each piece with pointed ends and median portion with pair of hooks; two large lateral amphids present.Esophagus divided into two parts; anterior esophagus (corpus) with distal bulb muscular, posterior esophagus (isthmus), ending in nonvalvulated bulb; nerve ring encircling isthmus; excretory pore anterior to esophageal bulb.Male with nine pairs of caudal papillae; spicules unequal, only larger (left) one narrower in middle portion, lanceolate; gubernaculum absent.Female monodelphic, ovoviviparous with vulva close to anus.
Etymology: The name of the genus comes from the greek "acanth/o," meaning spine or thorn, referring to the presence of spines on the cephalic end of the nematode.Jesus and Melo n. gen., n. sp.

General description
Medium-sized nematodes, body cylindrical, tapering to both extremities.Males and females similar in length without lateral alae.Esophagus divided into defined corpus, elongated isthmus, and a bulb.Nerve ring situated in middle of esophagus, at beginning of isthmus; deirids small, located just below nerve ring.Excretory pore anterior to esophageal bulb, opening surrounded by radial cuticle striations; pore opening into large chamber with transverse striations.Posterior end with short, pointed tail, ventrally curved in both sexes.
ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN, 2012), details of the new species have been submitted to ZooBank.The Life Science Identifier (LSID) for Acanthoatractis xinguensis n. gen., n. sp.Is urn: lsid: zoobank.org:act: ABE15A5D-A805-49BE-A544-DCBBDBA20EF6.
Etymology: The species was named after the Xingu River, the type locality.Jesus et al. (2022) proposed an identification key for Atractidae and listed 27 valid genera.Among these genera, Labeonema Puylaert, was included; however, molecular analyses indicated that the phylogenetic position of Labeonema is within Cosmocercidae Railliet, 1914(see Saito et al., 2021).Therefore, in this study, we will consider 26 valid genera, listed in a table that includes type species, host, and biogeographic occurrence of Atractidae genera (see Table 1).

Discussion
We assign the specimens described here to Atractidae because the esophagus is divided into two distinct portions: an anterior, cylindrical portion (corpus) and a posterior, elongated portion (isthmus) ending in a bulb.Males lack a precloacal sucker in the caudal region and possess two unequal spicules.Females are monodelphic and ovoviviparous, with the vulva located in the posterior region of the body, near the anus (Anderson et al., 2009).
The number and morphology of lips and the presence/absence of sclerotized structures such as spines and hooks are the main characteristics that differentiate genera within the Atractidae (Jesus et al., 2022).Based on these characteristics, Atractidae can be divided into two groups of species; the first group comprises genera whose lips have associated sclerotized structures (hooks and/or spines), including Klossinemella, Orientatractis, Crossocephalus, Paraorientatractis, Cobboldina, and Grassenema.The second group includes genera without sclerotized structures at the cephalic end, namely Nouvelnema, with the oral opening surrounded by two lips, Buckleyatractis, Labiduris, Paratractis, Pneumoatractis, and Pseudocyrtosomum with three lips; Cyrtosomum, Fitzsimmonsnema, Leiperenia, Monhysterides, Podocnematractis, Probstmayria, Pseudatractis, Rhinoceronema, and Rhinoclemmysnema, which have six lips; Vogtnema with four lips; and Hippopotamenema has the oral opening directed ventrally and lacks lips.Thus, Acanthoatractis n. gen., n. sp.belongs to the first group of genera in which the oral aperture has sclerotized structures.
Acanthoatractis n. gen.has eight wrench-shaped sclerotized structures around the mouth.Klossinemella and Orientatractis differ from the new genus by the number and shape of sclerotized structures around the mouth.Klossinemella has an oral opening surrounded by two bilobed formations (dorsal and ventral) resembling lips, with eight Y-shaped sclerotized structures, two subdorsal pairs, two laterodorsal pairs, two subventral pairs, and two lateroventral pairs.Additionally, a single horn is present between each subventral pair, and every two pairs have two small sublateral processes resembling papillae, sclerotized and oriented posteriorly to the amphids.Orientatractis species have six lips; each submedian lip has a well-sclerotized, recurved, pointed, bicornate chitinous piece and a single median spine.Furthermore, all species of Klossinemella and Orientatractis have a gubernaculum (Moravec and Thatcher, 1997;Jesus et al., 2023), whereas Acanthoatractis n. gen.lacks a gubernaculum.
The new genus lacks papillae at the cephalic end, and the oral opening has two sclerotized pieces, each with a pair of median hooks (one dorsal and one ventral).Crossocephalus, described in equines and rhinoceros, can be easily distinguished from the new genus Acanthoatractis by its numerous papillae at the cephalic end.Moreover, Crossocephalus has three lips (one dorsal and two submedian), each lip with a pair of hooks, and the anterior end of the esophagus with three pairs of eversible pectinate blades.The excretory pore is post-bulbar, and the esophageal bulb has valves (Berenger, 1947;York and Southwell, 1920), while in Acanthoatractis n. gen. the excretory pore is pre-bulbar, and the esophageal bulb does not have valves.
In Acanthoatractis n. gen., lips are absent, displaying eight pairs of spines (two subdorsal pairs, two dorsolateral pairs, two subventral pairs, and two ventrolateral pairs), and spines are absent posterior to the amphid pores.In contrast, Paraorientatractis from freshwater turtles has an oral opening with four submedian lips, each lip bearing a pair of recurved spines fused at the base and a single median spine near the distal margin, along with a pair of smaller spines posterior to the amphids.Additionally, Paraorientatractis has cuticular projections along the dorsal surface of the body (Gibbons et al., 1997), while Acanthoatractis n. gen.has a smooth cuticle.
Cobboldina found in hippopotami (Mammalia: Hippopotamidae), has a mouth surrounded by a cuticular sheath with a triangular median extension in dorsal and ventral positions (Leiper, 1911;Mondal and Manna, 2012) and differs from the new genus by the absence of hooks or spines in the mouth.
Finally, the new taxon differs by having the cephalic end surrounded by 16 pairs of spines distributed in a circle in subdorsal, dorsolateral, subventral, and ventrolateral positions.Grassenema, described in hiracoids (Mammalia: Hyracoidea), has the cephalic end surrounded by 12 pairs of cuticular spines arranged in two circles: the anterior circle with four pairs in subdorsal and subventral positions, and the posterior circle with eight pairs in subdorsal, subventral, and sublateral positions.Additionally, Grassenema possesses a buccal capsule and pharynx (Saito et al., 2021), structures that are absent in Acanthoatractis n. gen.
Thus, based on these morphological characters, the nematodes described here represent the 27th genus among atractids and the seventh report of this family in the P. unifilis species.

Table 1
Genus, type-species, host and Biogeographic occurrence of Atractidae (26 valid genera are included here and the new genus) around the world.